The long term goal of this project is to develop therapeutics that will result in the elimination of human immunodeficiency virus. The objective of this particular application, which represents a first step toward the attainment of our long term goal, is the development of Trojan horse inhibitors (THIs) that are activated by HIV protease and serve to kill the cells in which the HIV is attempting to replicate. The central hypothesis is that elimination of the cells in which the virus is activated can prevent viral replication. The first generation THI is a protein consisting of four parts: a cellular penetration sequence, a membrane-disruption peptide, a masking helix that blocks the action of the membrane-disruption peptide and a HIV protease cleavage site between the masking helix and the membrane- disruption peptide. The cellular penetration sequence promotes cellular uptake. In the absence of HIV, nothing happens. In the presence of HIV, the peptide is cleaved by HIV protease releasing the membrane-disruption peptide, the peptide disrupts membrane function leading to cell death and virus is not produced. The central hypothesis will be tested and the overall objectives of this application accomplished by pursuing the following specific aims: 1. Determine the in vitro functionality of THIs. Several important aspects of the functionality of the first and subsequent generation THIs can be tested in vitro including correct HIV protease cleavage, HIV protease activation and membrane disruption 2. Investigate the ability of the first generation THI to kill HIV infected cells and reduce virus production in tissue culture. THI should function in tissue culture to kill HIV infected cells and spare non-infected cells. 3. Design the second and subsequent generations of THI. The THI consists of four elements: a peptide uptake sequence, a membrane-disruptive peptide, a masking helix and the HIV protease site. These will be rationally optimized for function using knowledge-based design and the results of in vitro and tissue culture testing. The main innovation of this project is to combine the use of a general Trojan horse strategy (introducing a benign substance that is activated by a virus enzyme) with a biophysical killing mechanism that relies on membrane disruption. Trojan horse inhibitors have the potential to stop viral production and so could provide a route to a cure for AIDS. AIDS has killed 25 million people since being recognized as an epidemic in 1981. PUBLIC HEALTH RELEVANCE: There are almost 43 million people currently living with HIV/AIDS. There were 3.1 million deaths from AIDS and 4.6 million new infections in 2005. AIDS is a major health care problem worldwide. The proposed research is significant because it concerns the development of therapeutics that could lead to the development of a cure for AIDS.